Pancreatic beta cells persistently infected with coxsackievirus B4 are targets of NK cell-mediated cytolytic activity


It has been suggested that the persistence of coxsackieviruses-B (CV-B) in pancreatic beta cells plays a role in the pathogenesis of type 1 diabetes (T1D). Yet, immunological effectors, especially natural killer (NK) cells, are supposed to clear virus-infected cells. Therefore, an evaluation of the response of NK cells to pancreatic beta cells persistently infected with CV-B4 was conducted. A persistent CV-B4 infection was established in 1.1B4 pancreatic beta cells. Infectious particles were found in supernatants throughout the culture period. The proportion of cells containing viral protein VP1 was low (< 5%), although a large proportion of cells harbored viral RNA (around 50%), whilst cell viability was preserved. HLA class I cell surface expression was downregulated in persistently infected cultures, but HLA class I mRNA levels were unchanged in comparison with mock-infected cells. The cytolytic activities of IL-2-activated non-adherent peripheral blood mononuclear cells (PBMCs) and of NK cells were higher towards persistently infected cells than towards mock-infected cells, as assessed by an LDH release assay. Impaired cytolytic activity of IL-2-activated non-adherent PBMCs from patients with T1D towards infected beta cells was observed. In conclusion, pancreatic beta cells persistently infected with CV-B4 can be lysed by NK cells, implying that impaired cytolytic activity of these effector cells may play a role in the persistence of CV-B in the host and thus in the viral pathogenesis of T1D.

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  1. 1.

    Zell R, Delwart E, Gorbalenya AE, Hovi T, King AMQ, Knowles NJ, Lindberg AM, Pallansch MA, Palmenberg AC, Reuter G, Simmonds P, Skern T, Stanway G, Yamashita TIRC (2017) ICTV virus taxonomy profile: Picornaviridae. J Gen Virol 98:2421–2422

  2. 2.

    Hober D, Alidjinou EK (2013) Enteroviral pathogenesis of type 1 diabetes: queries and answers. Curr Opin Infect Dis 26:263–269

  3. 3.

    Hober D, Sauter P (2010) Pathogenesis of type 1 diabetes mellitus: interplay between enterovirus and host. Nat Rev Endocrinol 6:279–289

  4. 4.

    Oikarinen S, Martiskainen M, Tauriainen S et al (2011) Enterovirus RNA in blood is linked to the development of type 1 diabetes. Diabetes 60:276–279

  5. 5.

    Oikarinen M, Tauriainen S, Oikarinen S et al (2012) Type 1 diabetes is associated with enterovirus infection in gut mucosa. Diabetes 61:687–691

  6. 6.

    Yeung W-CG, Rawlinson WD, Craig ME (2011) Enterovirus infection and type 1 diabetes mellitus: systematic review and meta-analysis of observational molecular studies. BMJ 342:d35–d35

  7. 7.

    Alidjinou EK, Sané F, Engelmann I et al (2014) Enterovirus persistence as a mechanism in the pathogenesis of type 1 diabetes. Discov Med 18:273–282

  8. 8.

    Krogvold L, Edwin B, Buanes T et al (2015) Detection of a low-grade enteroviral infection in the islets of langerhans of living patients newly diagnosed with type 1 diabetes. Diabetes 64:1682–1687

  9. 9.

    Alidjinou EK, Chehadeh W, Weill J et al (2015) Monocytes of patients with type 1 diabetes harbour enterovirus RNA. Eur J Clin Investig 45:918–924

  10. 10.

    Yin H, Berg A-K, Tuvemo T, Frisk G (2002) Enterovirus RNA is found in peripheral blood mononuclear cells in a majority of type 1 diabetic children at onset. Diabetes 51:1964–1971

  11. 11.

    Sane F, Caloone D, Gmyr V et al (2013) Coxsackievirus B4 can infect human pancreas ductal cells and persist in ductal-like cell cultures which results in inhibition of Pdx1 expression and disturbed formation of islet-like cell aggregates. Cell Mol Life Sci 70:4169–4180

  12. 12.

    Alidjinou EK, Engelmann I, Bossu J et al (2017) Persistence of Coxsackievirus B4 in pancreatic ductal-like cells results in cellular and viral changes. Virulence 8:1229–1244

  13. 13.

    Chehadeh W, Kerr-Conte J, Pattou F et al (2000) Persistent infection of human pancreatic islets by coxsackievirus B is associated with alpha interferon synthesis in beta cells. J Virol 74:10153–10164

  14. 14.

    Richardson SJ, Morgan NG, Foulis AK (2014) Pancreatic pathology in type 1 diabetes mellitus. Endocr Pathol 25:80–92

  15. 15.

    Warren H, Smyth M (1999) NK cells and apoptosis. Immunol Cell Biol 77:64–75

  16. 16.

    Seliger B, Ritz U, Ferrone S (2006) Molecular mechanisms of HLA class I antigen abnormalities following viral infection and transformation. Int J Cancer 118:129–138

  17. 17.

    Smyth MJ, Cretney E, Kelly JM et al (2005) Activation of NK cell cytotoxicity. Mol Immunol 42:501–510

  18. 18.

    Vitale C, Chiossone L, Morreale G et al (2005) Human natural killer cells undergoing in vivo differentiation after allogeneic bone marrow transplantation: analysis of the surface expression and function of activating NK receptors. Mol Immunol 42:405–411

  19. 19.

    Dotta F, Censini S, van Halteren AGS et al (2007) Coxsackie B4 virus infection of beta cells and natural killer cell insulitis in recent-onset type 1 diabetic patients. Proc Natl Acad Sci USA 104:5115–5120

  20. 20.

    Baba M, Hasegawa H, Nakayabu M et al (1993) Cytolytic activity of natural killer cells and lymphokine activated killer cells against hepatitis A virus infected fibroblasts. J Clin Lab Immunol 40:47–60

  21. 21.

    Biron CA, Nguyen KB, Pien GC et al (1999) Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu Rev Immunol 17:189–220

  22. 22.

    Godeny EK, Gauntt CJ (1986) Involvement of natural killer cells in coxsackievirus B3-induced murine myocarditis. J Immunol 137:1695–1702

  23. 23.

    Godeny EK, Gauntt CJ (1987) Murine natural killer cells limit coxsackievirus B3 replication. J Immunol 139:913–918

  24. 24.

    Hühn MH, Hultcrantz M, Lind K, Ljunggren HG, Malmberg KJF-TM (2008) IFN-γ production dominates the early human natural killer cell response to Coxsackievirus infection. Cell Microbiol 10:426–436

  25. 25.

    Alidjinou EK, Sané F, Engelmann I, Hober D (2013) Serum-dependent enhancement of Coxsackievirus B4-induced production of IFNα, IL-6 and TNFα by peripheral blood mononuclear cells. J Mol Biol 425:5020–5031

  26. 26.

    Alidjinou EK, Sané F, Trauet J et al (2015) Coxsackievirus B4 can infect human peripheral blood-derived macrophages. Viruses 7:6067–6079

  27. 27.

    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408

  28. 28.

    Marca V, Gianchecchi E, Fierabracci A (2018) Type 1 diabetes and its multi-factorial pathogenesis: the putative role of NK cells. Int J Mol Sci.

  29. 29.

    Cornell CT, Kiosses WB, Harkins S, Whitton JL (2007) Coxsackievirus B3 proteins directionally complement each other to downregulate surface major histocompatibility complex class I. J Virol 81:6785–6797

  30. 30.

    Campbell IL, Bizilj K, Colman PG et al (1986) Interferon-gamma induces the expression of HLA-A, B, C but not HLA-DR on human pancreatic beta-cells. J Clin Endocr Metab 62:1101–1109

  31. 31.

    Rodríguez T, Méndez R, Del Campo A et al (2007) Distinct mechanisms of loss of IFN-gamma mediated HLA class I inducibility in two melanoma cell lines. BMC Cancer 7:34.

  32. 32.

    Cao J, Brouwer NJ, Jordanova ES et al (2018) HLA class I antigen expression in conjunctival melanoma is not associated with PD-L1/PD-1 status. Investig Ophthalmol Vis Sci 59:1005–1015

  33. 33.

    Roivainen M, Rasilainen S, Ylipaasto P et al (2000) Mechanisms of coxsackievirus-induced damage to human pancreatic beta-cells. J Clin Endocr Metab 85:432–440

  34. 34.

    Vuorinen T, Nikolakaros G, Simell O et al (1992) Mumps and Coxsackie B3 virus infection of human fetal pancreatic islet-like cell clusters. Pancreas 7:460–464

  35. 35.

    Yoon JW, Onodera T, Jenson AB, Notkins AL (1978) Virus-induced diabetes mellitus. XI. Replication of coxsackie B3 virus in human pancreatic beta cell cultures. Diabetes 27:778–781

  36. 36.

    McCluskey JT, Hamid M, Guo-Parke H et al (2011) Development and functional characterization of insulin-releasing human pancreatic beta cell lines produced by electrofusion. J Biol Chem 286:21982–21992

  37. 37.

    Benkahla MA, Alidjinou EK, Sane F et al (2018) Fluoxetine can inhibit coxsackievirus-B4 E2 in vitro and in vivo. Antiviral Res 159:130–133

  38. 38.

    Alidjinou EK, Sané F, Bertin A et al (2015) Persistent infection of human pancreatic cells with Coxsackievirus B4 is cured by fluoxetine. Antiviral Res 116:51–54

  39. 39.

    Heim A, Canu A, Kirschner P et al (1992) Synergistic interaction of interferon-beta and interferon-gamma in coxsackievirus B3-infected carrier cultures of human myocardial fibroblasts. J Infect Dis 166:958–965

  40. 40.

    Heim A, Brehm C, Stille-Siegener M et al (1995) Cultured human myocardial fibroblasts of pediatric origin: natural human interferon-alpha is more effective than recombinant interferon-alpha 2a in carrier-state coxsackievirus B3 replication. J Mol Cell Cardiol 27:2199–2208

  41. 41.

    Pinkert S, Klingel K, Lindig V et al (2011) Virus-host coevolution in a persistently coxsackievirus B3-infected cardiomyocyte cell line. J Virol 85:13409–13419

  42. 42.

    Richardson SJ, Willcox A, Bone AJ et al (2009) The prevalence of enteroviral capsid protein vp1 immunostaining in pancreatic islets in human type 1 diabetes. Diabetologia 52:1143–1151

  43. 43.

    Pujol-Borrell R, Todd I, Doshi M et al (1986) Differential expression and regulation of MHC products in the endocrine and exocrine cells of the human pancreas. Clin Exp Immunol 65:128–139

  44. 44.

    Deitz SB, Dodd DA, Cooper S et al (2000) MHC I-dependent antigen presentation is inhibited by poliovirus protein 3A. Proc Natl Acad Sci USA 97:13790–13795

  45. 45.

    Moffat K, Howell G, Knox C et al (2005) Effects of foot-and-mouth disease virus nonstructural proteins on the structure and function of the early secretory pathway: 2BC but not 3A blocks endoplasmic reticulum-to-Golgi transport. J Virol 79:4382–4395

  46. 46.

    Kirkegaard K, Taylor MP, Jackson WT (2004) Cellular autophagy: surrender, avoidance and subversion by microorganisms. Nat Rev Microbiol 2:301–314

  47. 47.

    de Jong AS, Visch H-J, de Mattia F et al (2006) The coxsackievirus 2B protein increases efflux of ions from the endoplasmic reticulum and Golgi, thereby inhibiting protein trafficking through the Golgi. J Biol Chem 281:14144–14150

  48. 48.

    Cornell CT, Kiosses WB, Harkins S, Whitton JL (2006) Inhibition of protein trafficking by coxsackievirus b3: multiple viral proteins target a single organelle. J Virol 80:6637–6647

  49. 49.

    Salvesen GS, Dixit VM (1997) Caspases: intracellular signaling by proteolysis. Cell 91:443–446

  50. 50.

    Li ZM, Liu ZC, Guan ZZ et al (2004) Inhibition of DNA primase and induction of apoptosis by 3,3′-diethyl-9-methylthia-carbocyanine iodide in hepatocellular carcinoma BEL-7402 cells. World J Gastroenterol 10:514–520

  51. 51.

    Vives-Pi M, Rodríguez-Fernández S, Pujol-Autonell I (2015) How apoptotic β-cells direct immune response to tolerance or to autoimmune diabetes: a review. Apoptosis 20:263–272

  52. 52.

    Mathis D, Vence L, Benoist C (2001) Beta-cell death during progression to diabetes. Nature 414:792–798

  53. 53.

    O’Brien BA, Geng X, Orteu CH et al (2006) A deficiency in the in vivo clearance of apoptotic cells is a feature of the NOD mouse. J Autoimmun 26:104–115

  54. 54.

    Eizirik DL, Grieco FA (2012) On the immense variety and complexity of circumstances conditioning pancreatic-cell apoptosis in type 1 diabetes. Diabetes 61:1661–1663

  55. 55.

    Wilson RG, Anderson J, Shenton BK et al (1986) Natural killer cells in insulin dependent diabetes mellitus. Br Med J 293:244

  56. 56.

    Hussain MJ, Alviggi L, Millward BA et al (1987) Evidence that the reduced number of natural killer cells in type 1 (insulin-dependent) diabetes may be genetically determined. Diabetologia 30:907–911

  57. 57.

    Negishi K, Waldeck N, Chandy G et al (1986) Natural killer cell and islet killer cell activities in type 1 (insulin-dependent) diabetes. Diabetologia 29:352–357

  58. 58.

    Lorini R, Moretta A, Valtorta A et al (1994) Cytotoxic activity in children with insulin-dependent diabetes mellitus. Diabetes Res Clin Pract 23:37–42

  59. 59.

    Qin H, Lee IF, Panagiotopoulos C et al (2011) Natural killer cells from children with type 1 diabetes have defects in NKG2D-dependent function and signaling. Diabetes 60:857–866

  60. 60.

    Rodacki M, Svoren B, Butty V et al (2007) Altered natural killer cells in type 1 diabetic patients. Diabetes 56:177–185

  61. 61.

    Hofmann P, Schmidtke M, Stelzner A, Gemsa D (2001) Suppression of proinflammatory cytokines and induction of IL-10 in human monocytes after coxsackievirus B3 infection. J Med Virol 64:487–498

  62. 62.

    Ylipaasto P, Klingel K, Lindberg AM et al (2004) Enterovirus infection in human pancreatic islet cells, islet tropism in vivo and receptor involvement in cultured islet beta cells. Diabetologia 47:225–239

  63. 63.

    Schulte BM, Bakkers J, Lanke KHW et al (2010) Detection of enterovirus RNA in peripheral blood mononuclear cells of type 1 diabetic patients beyond the stage of acute infection. Viral Immunol 23:99–104

  64. 64.

    Willcox A, Richardson SJ, Bone AJ et al (2011) Immunohistochemical analysis of the relationship between islet cell proliferation and the production of the enteroviral capsid protein, VP1, in the islets of patients with recent-onset type 1 diabetes. Diabetologia 54:2417–2420

  65. 65.

    Lima JF, Oliveira LMS, Pereira NZ et al (2017) Polyfunctional natural killer cells with a low activation profile in response to Toll-like receptor 3 activation in HIV-1-exposed seronegative subjects. Sci Rep 7:524.

  66. 66.

    Flodström M, Maday A, Balakrishna D et al (2002) Target cell defense prevents the development of diabetes after viral infection. Nat Immunol 3:373–382

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This work was supported by Ministere de l’Education Nationale de la Recherche et de la Technologie, Universite Lille 2 (Equipe d’accueil 3610), Centre Hospitalier Regional et Universitaire de Lille, and by EU FP7 (GA-261441-PEVNET: Persistent virus infection as a cause of pathogenic inlammation in type 1 diabetes—an innovative research program of biobanks and expertise). M. P. N was supported by a “CABRI 2016” scholarship of Universite Lille 2 and a “Programme Eifel 2017” scholarship of Ministere des Afaires etrangeres et du Developpement International de la Republique Francaise. Funding was supported by Campus France (EIFFELDOCTORAT 2017/n°P714914K). The authors thank Dr Sarah Richardson (Exeter, UK) for helpful discussion. The authors thank Dr Adrian J. F. Luty for reading the manuscript.

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Nekoua, M.P., Bertin, A., Sane, F. et al. Pancreatic beta cells persistently infected with coxsackievirus B4 are targets of NK cell-mediated cytolytic activity. Cell. Mol. Life Sci. 77, 179–194 (2020) doi:10.1007/s00018-019-03168-4

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  • Enterovirus
  • Persistence
  • HLA class I
  • Type 1 diabetes
  • LDH assay